Top PDF Human immunodeficiency virus integration in a cell-free system.

Human immunodeficiency virus integration in a cell-free system.

Human immunodeficiency virus integration in a cell-free system.

To determine whether any nucleotides or other small molecules were required as cofactors for HIV integration, the crude cytoplasmic extract from infected cells was fractionated by gel ex[r]

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Primary Cell Model for Activation-Inducible Human Immunodeficiency Virus

Primary Cell Model for Activation-Inducible Human Immunodeficiency Virus

Impact of NF- ␬ B on HIV activation. Induction of the viral LTR is tightly regulated in our in vitro model system. We therefore determined if our model could be used to assess the importance of various LTR-binding transcription factors on the reactivation of HIV. Since replication-incompetent re- porter viruses are used in this assay, the effect of specific LTR mutations on viral expression levels can be clearly determined in a single round of infection. This approach allows us to focus on how the induced mutations affect reactivation without the complications caused by additional effects on replication. NF- ␬ B has long been established as an important regulator of HIV expression (36). Therefore, we chose to determine the level of involvement of NF- ␬ B response elements in HIV re- activation in our primary cell model. The NF- ␬ B sites within the enhancer region of the 3 ⬘ LTR of NLEGFPLuc were abol- ished by mutating three nucleotides within each NF- ␬ B re- sponse element to previously described sequences (36) that inhibit NF- ␬ B binding (Fig. 5A). The mutations were induced in the U3 region of the 3 ⬘ LTR because this region becomes the promoter of the virus after reverse transcription is com- pleted. To confirm that NF- ␬ B binding was abolished, we conducted EMSAs using nuclear extracts from CD4 ⫹ CD8 ⫹ thymocytes cultured in vitro for 10 days. As expected, costimu- lation led to an increase in protein binding to a probe spanning the NF- ␬ B response elements (Fig. 5B, compare lanes 2 and 3). Antibody supershifts confirmed that these DNA binding complexes contained NF- ␬ B subunits p65 (Fig. 5B, lane 4) and p50 (lane 5). However, the NF- ␬ B response element mutations
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Effect of Latent Human Immunodeficiency Virus Infection on Cell Surface Phenotype

Effect of Latent Human Immunodeficiency Virus Infection on Cell Surface Phenotype

anti-CD3 stimulation and can alter gene expression of proteins known to be crucial to HIV replication (32). Similarly, other studies have demonstrated that the viral protein tat, in addition to increasing HIV transcription, also transactivates cellular interleukin 2 gene expression (34, 36). While it is not known if HIV causes changes in cellular phenotype prior to the transi- tion into latency, it is possible that infection induces a change in cellular physiology that is not reversed when the infected cell enters a quiescent state. Alternatively, the coincident loss of viral gene transcription during cellular quiescence may cause these phenotypic changes to revert and the cell would then appear similar to an uninfected T cell. Due to the extremely low levels of latent virus in vivo, characterization of the latent reservoir at the cellular level has been difficult. In the SCID-hu system, latent HIV infection is generated at high frequency and the use of reporter viruses allows identification of produc- tively infected cells (5, 20). This system thus provides a pow- erful model to assess the properties of latently infected T cells and the effects of virus reactivation on the infected cell. Herein we demonstrate that latent infection does not significantly alter the normal phenotype of human thymocytes or peripheral blood lymphocytes. However, upon stimulation of the latently infected cell, viral proteins are produced and the characteristic, virally mediated manifestations of CD4 and MHC-I down- regulation are observed. These data suggest that the latently infected cell will appear normal to the immune system and thus escape immune clearance.
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Human Immunodeficiency Virus Persistence and Production in T-Cell Development

Human Immunodeficiency Virus Persistence and Production in T-Cell Development

DNA isolation and PCR. DNA was isolated from in vitro-infected cultures at 18 h postinfection by phenol-chloroform extraction and ethanol precipitation as described previously (27). For samples collected from SCID-hu thymocyte sub- sets, simultaneous extraction of RNA and DNA was performed using TRIzol (Gibco/BRL-Life Technologies), following the manufacturer’s protocol. Quan- titative PCR was performed using an ABI Prism 7700 sequence detection system in a 25-␮l reaction volume for 40 cycles between 95°C for 15 seconds and 60°C for 1 min, using the components in the TaqMan core reagent kit (Applied Biosystems, Foster City, CA). Proviral burden was measured using the primer pair SR1/AA55, which binds within the R and U5 regions of the long terminal repeat (LTR). This same pair was used to detect nucleotides synthesized at the initiation of reverse transcription. The SR1/661 primer pair spans the primer binding site and measures the completion of reverse transcription. The primers SR1 (5⬘-CAAGTAGTGTGTGCCCGTCTGT-3⬘), AA55 (5⬘-CTGCTAGAGAT TTTCCACACTGAC-3⬘), and 661 (5⬘-CCTGCGTCGAGAGAGCTCCTCTG G-3 ⬘ ) were used along with the fluorogenic probe ZXF (5 ⬘ -TGTGACTCTGGT AACTAGAGATCCCTCAGACCC-3 ⬘ ), modified with 6-carboxyfluorescein reporter dye on the 5⬘ end and 6-carboxytetramethylrhodamine quencher dye on the 3 ⬘ end. To control for the amount of cellular DNA per reaction, the following primers and probe were used to amplify a portion of the human beta-globin gene: forward primer BGF1 (5⬘-CAACCTCAAACAGACACCATGG-3⬘), reverse primer BGR1 (5⬘-TCCACGTTCACCTTGCCC-3⬘), and the fluorogenic probe modified as described above (5 ⬘ -CTCCTGAGGAGAAGTCTGCCGTTACTG CC-3 ⬘ ).
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Inducible human immunodeficiency virus type 1 packaging cell lines.

Inducible human immunodeficiency virus type 1 packaging cell lines.

To confirm vector virus transfer, drug-resistant HeLaT4 tar- get cells were cloned and the genomic DNA isolated from them was subjected to Southern blotting. Genomic DNA was digested with SacI and probed with the nef, neo, and gpt genes to detect 3.8-, 4.5-, and 3.4-kbp fragments indicating the pres- ence of the HVP, HSN, and HIV-gpt vector, respectively (Fig. 5). The results demonstrated that all puromycin-, neomycin-, and GPT-resistant cell clones contained HVP, HSN, and HIV- gpt vector proviruses, respectively. It should be noted that the additional bands observed with HVP-infected samples corre- spond to junction fragments between the 59 LTR and genomic DNA at the site of integration. These bands were observed because the nef probe also has homology to the 59 LTR up- stream of the SacI site. As expected, since the different provi- ruses integrate into different sites, these additional bands vary in size. Also, one of the neomycin-resistant HSN-infected cell clones showed a smaller provirus fragment after SacI digestion. This could be due to mutation during vector virus replication, which occurs at high frequency for retroviruses (37). These data indicate that both the packaging cell line and the Env- expressing cell line could effectively propagate defective HIV vector.
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Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.
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Mouse T cells restrict replication of human immunodeficiency virus at the level of integration

Mouse T cells restrict replication of human immunodeficiency virus at the level of integration

Carefully controlled virion fusion and real-time PCR anal- yses showed that reverse transcription per fused HIV-1 particle is at least as efficient in T-cells from mice as in their human counterparts. Furthermore, relative levels of 2-LTR circles were comparable or in some instances even markedly elevated in mouse T-cells (Figs. 3D, 8A), dem- onstrating transfer of the pre-integration complex into the nucleus. In contrast, Baumann et al. [20] reported reduced efficiencies at both of these replication steps in the identi- cal mouse T-cell lines used in our study. The reason for these discrepancies is currently unclear. In this report [20] we noted several differences to our study, including (i) a side-by-side comparison of infected mouse T-cells only with mouse NIH3T3 fibroblasts, but not human T-cells, (ii) a lack of correlative evaluations of consecutive replica- tion steps, (iii) a lack of control for residual proviral plas- mid contamination in virus inocula, and (iv) quantification of HIV-1 cDNA species without a PCR- based normalization for corresponding cell equivalents. In line with our observations, Tsurutani et al. [21] found that reverse transcription proceeds normally in mouse T- cells and that levels of 2-LTR circles were similar in the cross-species comparison following an HIV-1 IN wt infec- tion. Using a cassette ligation-mediated PCR these authors noted a qualitative reduction in integrants in primary lymphocytes from mice. However, based on 2- to 8-fold lower relative 2-LTR circle levels for infections with an integrase-defective HIV-1, Tsurutani et al. located the restriction exclusively at the level of nuclear import in mouse cells. Conceivably, the abundance of 2-LTR circles can be affected by a large number of parameters including
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Antigenic Properties of the Human Immunodeficiency Virus Transmembrane Glycoprotein during Cell-Cell Fusion

Antigenic Properties of the Human Immunodeficiency Virus Transmembrane Glycoprotein during Cell-Cell Fusion

It is noteworthy that MAbs against cluster I and cluster II epitopes fail to neutralize HIV infection (23, 41, 46, 55), even though they bind sequences in or near HR1 and HR2. It was previously speculated that the anti-cluster I and anti-cluster II MAbs are incapable of preventing fusion because their cognate epitopes are presented mainly on “dead spikes” of six-helix bundles (15) that appear only after membrane fusion has be- gun. This scenario now seems unlikely given our results show- ing that MAb binding clearly precedes, rather than follows, fusion (Fig. 1). Accordingly, we attempted to promote neutral- ization by mixing the MAbs with cells that were temperature arrested as soon as the cognate antigen was exposed. For example, 10-min cocultures were temperature arrested and, at the same low temperature, treated with the MAbs (Fig. 5A). Under these conditions, prehairpin structures form but do not fold into six-helix bundles (47). Thus, T20 strongly inhibited fusion when the system was returned to the fusogenic temper- ature of 37°C (Fig. 5A). In contrast, the MAbs failed to sup- press fusion (Fig. 5A), even though they reacted strongly with the arrested cells (data not shown). The MAbs also failed to block the fusion of Env cells that were triggered by sCD4, which were not subject to any spatial constraints on antibody binding that might be introduced by cell-cell contact (Fig. 5B). Taken together, these results could indicate that the structures recognized by the MAbs are mainly “off-pathway” and do not fold into active fusogenic structures. Such antigens, if present in sufficient numbers, might even act as decoys to lure the MAbs away from fusion-competent gp41 structures. However, this model is difficult to reconcile with the disappearance of MAb reactivity upon the initiation of fusion. Alternatively, it is possible that our cell-cell fusion system is highly efficient and therefore inherently resistant to interference by certain anti- gp41 antibodies. Such resistance may apply only to antibodies against cluster I or cluster II epitopes, since experiments in which we preincubated Env cells with 1.6 ␮M MAb 2F5 re-
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Integration Targeting by Avian Sarcoma-Leukosis Virus and Human Immunodeficiency Virus in the Chicken Genome

Integration Targeting by Avian Sarcoma-Leukosis Virus and Human Immunodeficiency Virus in the Chicken Genome

Comparing de novo integration and ERVs in the chicken genome. The distribution of de novo ASLV integration sites was quite different from that of related ERVs, suggesting mod- els for the forces determining which ERVs persist in the ge- nome. Both total chicken ERVs and the ASLV-related ERV subset have accumulated outside of TUs, and the minority of ERVs within TUs were typically in antisense orientation rela- tive to host cell transcription. For ERVs in the antisense ori- entation, the viral splicing and polyadenylation signals do not affect mRNA synthesis by the host gene, thereby minimizing the genetic damage of integration. Comparison of the de novo distribution of ASLV integration sites to related ERVs indi- cates that the present distribution of chicken ERVs relative to TUs was likely not determined by the initial integration tar- geting, but rather by selective pressures against gene disrup- tion. This trend has been noted previously for human ERVs (27), but the analysis of ASLV presented here provides the first case where the de novo pattern of integration was experimen- tally determined, allowing the observed biases in ERV distri- bution to be attributed to forces acting after integration.
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Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

In addition, in all three cell types used in this study peripheral blood lymphocytes, Mo-T, and MT-4, infection with wild-type HIV-1 mfA virions and integrase mutants resulted in the syn[r]

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A Sensitive, Quantitative Assay for Human Immunodeficiency Virus Type 1 Integration

A Sensitive, Quantitative Assay for Human Immunodeficiency Virus Type 1 Integration

Integration, an obligate step in retrovirus replication, is de- fined as the covalent insertion of viral DNA into the host cell genome. Human immunodeficiency virus type 1 (HIV-1) pro- viruses appear to contribute to viral persistence. This is most evident in treated patients, where the proviral load seems to be unaffected by combination antiretroviral therapy that clears viremia (10). To date, proviral DNA has proven impossible to eradicate without death of the transduced host cell. These results suggest that provirus may serve as a treatment-resistant reservoir of HIV-1. Quantitative studies of this reservoir are challenging because of the relative rarity of integration events in quiescent leukocytes. Assays for HIV-1 integration have been implemented using three main strategies, namely, inverse PCR (7), linker-primer PCR (37), and Alu PCR (2, 3, 5, 12, 34). We focus on and refine the latter method here.
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Cell-Free versus Cell-to-Cell Infection by Human Immunodeficiency Virus Type 1 and Human T-Lymphotropic Virus Type 1: Exploring the Link among Viral Source, Viral Trafficking, and Viral Replication

Cell-Free versus Cell-to-Cell Infection by Human Immunodeficiency Virus Type 1 and Human T-Lymphotropic Virus Type 1: Exploring the Link among Viral Source, Viral Trafficking, and Viral Replication

Both VS formation and filopodium-dependent stabilization involve cytoskeleton remodeling in infected cells. In the context of DC-to-T-cell transmission, HIV-1 triggers actin polymerization through the use of the formin 2 Rho-GTPase, CDC42, and Env, Nef, and Gag viral proteins (51). HTLV-1 alters actin polymeriza- tion through p8, which is responsible for increasing conduit for- mation (63), and through Tax by upregulating Gem (64). In HTLV-1-infected cells, Gem colocalizes with actin and strongly increases both formation of conjugates between infected and un- infected lymphocytes and viral transfer (64). In contrast to the case for HIV-1, CDC42 is not involved in HTLV-1-induced actin polymerization, although it interacts with Tax in CD4 ⫹ T lym- phocytes (65). Both retroviruses are also known to act on the microtubule network by inducing microtubule-organizing center (MTOC) polarization during VS formation (66, 67). MTOC po- larization is a hallmark of the immune synapse. Importantly, it occurs in the donor cell and not in the target cell in the case of the FIG 2 Infection after cell-cell contact: the viral synapse. The viral synapse is characterized by an intimate contact between the infected donor cell (left) and the target cell (right). The formation of the VS can be arbitrarily divided into 6 steps: 1, cell-cell contact is established through interactions between fusion- incompetent viral Env proteins (represented in yellow) and ICAM-1 on the donor cell side and viral receptor (represented in blue) and LFA-1 on the target cell; 2, adhesion leads to MTOC polarization and virion assembly at the cell-cell contact in the donor cell; 3, newly synthesized virions are released in the synaptic cleft; 4, polarized capture of virions by the target cell is driven by Env-receptor interaction; 5, captured virions are internalized through endocytosis in the target cell; and 6, Gag maturation in endosomes leads to Env-mediated viral fusion and release of viral capsids in the cytosol, allowing productive infection.
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Rapid induction of apoptosis by cell-to-cell transmission of human immunodeficiency virus type 1.

Rapid induction of apoptosis by cell-to-cell transmission of human immunodeficiency virus type 1.

CD4 1 T-cell depletion remains a key unexplained charac- teristic of acquired immunodeficiency, which predisposes the patient to lethal infectious and neoplastic complications of AIDS and for which no therapy is effective. Programmed cell death has been postulated as a potential mechanism for CD4 1 T-cell loss in HIV-infected persons (15, 33, 43, 52) because lymphocytes from infected persons undergo accelerated apop- tosis after cultivation in vitro. It is of interest that no acceler- ated apoptosis was detected in lymphocytes cultured from HIV-infected chimpanzees, animals which become viremic but show no CD4 T-cell decline and no disease progression after HIV infection (16, 48). No correlation between susceptibility to apoptosis in vitro and CD4 decline or AIDS disease pro- gression has been observed (34), and apoptosis has been ob- served in peripheral blood mononuclear cells cultured from patients with chronic illnesses other than HIV infection, in- cluding leukemia and systemic lupus erythematosus (9, 21, 46), suggesting that accelerated programmed cell death ex vivo may represent a nonspecific defect in peripheral blood mononu- clear cell longevity or a result of imperfect cultivation condi- tions and not a specific pathologic consequence of HIV infec- tion. Recently, apoptotic cells have been identified in lymph nodes of HIV-infected children and simian immunodeficiency virus-infected primates, primarily among bystander cells and not virus-infected cells (13). Our finding that infected cells efficiently and rapidly induce apoptosis following cell-to-cell transmission of HIV suggests apoptosis may contribute to pro- gressive lymphocyte depletion within organs such as lymph nodes, in which relatively few infected cells are present (44) but extensive cell-cell interactions are possible. In addition, direct T-cell killing has been inferred in explaining CD4 cell decline in HIV infection and the recovery of CD4 cells after treatment with HIV protease inhibitors (18, 54). In this regard, we have detected rapid induction of apoptosis after cocultiva- tion of peripheral blood mononuclear cells from uninfected donors with chronically infected H9 cells (unpublished data), demonstrating that HIV-associated apoptosis is efficiently in- duced in lymphocytes as well as in T-cell lines. By continuing to characterize the multiple mechanisms that result in the elimi- nation of CD4 1 cells, including apoptosis, we may identify novel approaches to prevent lymphocyte depletion and pro- long the disease-free period following the initial exposure to HIV.
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Impaired Accessory Cell Function in a Human Dendritic Cell Line after Human Immunodeficiency Virus Infection

Impaired Accessory Cell Function in a Human Dendritic Cell Line after Human Immunodeficiency Virus Infection

We generated human dendritic cell (DC) hybridoma cell lines by fusing HGPRT-deficient promonocytic U937 cells with immature DCs obtained by culturing peripheral blood monocytes with interleukin-4 (IL-4; 1,000 U/ml) and granulocyte-macrophage colony-stimulating factor (100 U/ml) for 7 days and mature DCs by treatment with tumor necrosis factor alpha (12.5 ␮ g/ml) for 3 days. Only one fusion with immature DCs was successful and yielded four cell lines—HB-1, HB-2, HB-3, and HB-9—with an overall fusion efficiency of 0.0015%. The cell lines were stable in long-term culture, displayed morphological features typical of DCs, and expressed distinct class I and class II molecules not present on U937 (A*031012, B*51011, Cw*0701, DRB3*01011 52, and DR5*01011). A representative cell line, HB-2, that expressed DC markers including CD83, CD80 and CD86 could be induced to produce IL-12 through CD40 stimulation. After human immuno- deficiency virus (HIV) infection, there was impairment of antigen-presenting cell (APC) function, which was manifested by an inability to stimulate allogeneic T-cell responses. There was no change in expression of major histocompatibility complex class I and class II antigens, CD83, CD40, CD4, CD11c, CD80, CD86, CD54, and CD58, or IL-12 production in the HIV-infected HB-2 cells. The HIV-infected HB-2 cells induced T-cell apoptosis in the cocultures. T-cell proliferation could be partially restored by using ddI, indinivir, and blocking anti-gp120 and anti-IL-10 antibodies. Our data suggest that there are multiple mechanisms that DCs use to inhibit T-cell responses in HIV-infected patients. The HB-2 cell line could be a useful model system to study APC function in HIV-infected DCs.
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Human Immunodeficiency Virus Integration Efficiency and Site Selection in Quiescent CD4+ T Cells

Human Immunodeficiency Virus Integration Efficiency and Site Selection in Quiescent CD4+ T Cells

A more detailed examination of the viral life cycle in quies- cent CD4 ⫹ T cells can shed more light on the nature of the block presented by quiescent CD4 ⫹ T cells. A series of recent studies looking at the different stages of the HIV life cycle in quiescent cells have further supported data from our earlier work (82, 83). More specifically, it has been shown that reverse transcription is inefficient in quiescent cells, generating full- length viral transcripts that are very labile (half-life of 1 day) but are integration competent (60, 84). However, whether there is integrated provirus in these cells has not been deter- mined. In another study, provirus can be found in resting cells of HIV-infected individuals, but this was attributed to previ- ously activated cell populations that returned to a resting state after stimulation, such as memory T cells (31, 35). The devel- opment of more-sensitive Alu PCR-based assays allowed for the detection of very low copy numbers of integrated HIV (2, 57). Several studies showed, using alternative methods of in- fection, such as spinoculation, that a latent infection can be established in quiescent CD4 ⫹ T cells (58, 73, 74). Our group undertook a comprehensive study and examined multiple stages of viral replication (entry, reverse transcription, integra- tion, viral gene transcription, and viral protein synthesis) in quiescent cells and compared them to those of stimulated cells. Quiescent CD4 ⫹ T cells were infected and then immediately stimulated to determine if this will rescue infection. Replica- tion in these cells stimulated immediately after infection was characterized by a long delay in reverse transcription and in- tegration compared to that in prestimulated T cells (78). Re- verse transcription was largely inefficient (30-fold less than stimulated cells), integration efficiency was slightly decreased (twofold) and protein expression was very poor. Thus, imme- diate stimulation failed to effectively rescue infection of quies- cent cells. Interestingly, their kinetics of infection was very similar to that of infected quiescent cells that remained un- stimulated. These data suggested that a major block of HIV * Corresponding author. Mailing address: 615 Charles E. Young
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Antigenic Properties of the Human Immunodeficiency Virus Envelope during Cell-Cell Fusion

Antigenic Properties of the Human Immunodeficiency Virus Envelope during Cell-Cell Fusion

Because of their unique structures, HIV envelope interme- diates have the potential to elicit distinct immune responses, possibly including broadly neutralizing antibodies. Recent ev- idence with either subunit or cell-based immunogens supports this concept (5, 17). One array of such epitopes is induced on gp120 by CD4 binding and is specific to the gp120-CD4 com- plex. Some of these epitopes comprise the coreceptor-binding domain and are being considered as potentially important tar- gets for antibodies to inhibit virus-mediated membrane fusion. However, despite antibody recognition of these epitopes on soluble gp120-CD4 complexes, it is unclear whether such re- activity occurs in the context of cell-cell or virus-cell membrane fusion. Monoclonal antibodies (MAbs) against conserved CD4-induced epitopes potently block soluble CD4 (sCD4)- activated fusion with target cells expressing coreceptor alone but have minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor (23). Other antibodies raised against gp120-CD4 complexes are ei- ther poorly neutralizing (5) or variably enhance or inhibit in- fection, depending on the assay conditions (18, 25). Therefore, the successful development of effective immunogens based on altered HIV envelope structures must consider the antigenic * Corresponding author. Mailing address: Institute of Human Vi-
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Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

coness Medical Center) approval. Specimens from patients with inflam- matory bowel disease were excluded. In all cases, samples for this study were taken adjacent to margins of resection in order to ensure the use of both macroscopically and microscopically normal tissues. Some of the materials were fixed in 4% paraformaldehyde and then embedded in par- affin for hematoxylin-eosin (H&E) staining to confirm a normal structure and tissue integrity. Nonfixed tissue samples were washed repeatedly and incubated for 15 min in R10 supplemented with 15 ␮ g/ml gentamicin (Life Technologies), 15 ␮ g/ml ciprofloxacin (Sigma-Aldrich), 120 U/ml nystatin (Sigma-Aldrich), and 25 ␮ g/ml amphotericin B (Sigma-Aldrich). The muscularis propria side of the tissue was marked with black glitter particles (American Crafts, Orem, UT) to distinguish the mucosal side from the muscularis propria side during the sealing stage. The tissue was divided into circular pieces 4 mm in diameter with a biopsy punch and transferred to a 24-well plate with 0.5 ml of R10 supplemented with 20 U/ml IL-2, 5 ␮g/ml insulin (Sigma-Aldrich), 5 ␮g/ml transferrin (Sigma- Aldrich), 5 ng/ml selenium (Sigma-Aldrich), 2 ng/ml epidermal growth factor (Life Technologies), 15 ␮ g/ml gentamicin, and 15 ␮ g/ml cipro- floxacin. The biopsy specimens were sealed with a 3% solution of agarose (Lonza, Walkersville, MD) in double-distilled water. The mucosal side was positioned against a cold glass slide, and the opposite glitter-coated exposed side of the biopsy specimen was covered in gel. After rapid solid- ification of the gel, the biopsy specimens were transferred to a 24-well plate containing the culture medium described above with the luminal side facing upwards, allowing the unsealed mucosa to be exposed to free virus or cell-associated virus. Cell-free replication-competent HIV-1 (R5) NL4.3-BaL-GFP or CellTrace Far Red (DDAO-SE)-stained human CD4 ⫹ T lymphocytes infected with HIV-1 (R5) NL4.3-BaL-GFP were intro- duced on top of the sealed explants under shaking conditions at 37°C for 2 days. The sealed explant tissue specimens were then washed with R10 and maintained in culture for 3 additional days at 37°C before explant cell isolation.
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Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.

Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.

FIG. 5. Cell-free plus-strand synthesis from non-PPT sites. DNA synthesis reactions were carried out for 40 min as described in Materials and Methods and the legends to Fig. 3 and 4. Plus-strand initiation sites are indicated with arrows to the left of each panel, and HIV-1 sequence positions are marked alongside the plus-strand sequence lanes (GATC) in the right margins. (A) Plus-strand prod- ucts generated from primer 4765 z pol RNA (Fig. 1). (B) Plus-strand products generated from primer 8000 z env RNA (Fig. 1). Lanes: 1, reactions with wild- type HIV-1 RT; 2, reactions with RNase H-minus HIV-1 RT (D443N); 3, 20-mer markers. (C) Conversion efficiencies for each initiation site. Percent conversion for each site was calculated from the molar quantity of an individual plus-strand product (determined from standards used in each blot [data not shown]) relative to the molar quantity of minus-strand DNA extended $18 nt beyond the initi- ation site giving rise to that product (Fig. 3 and data not shown). Calculations were made separately for each initiation site, thus normalizing for differences in the efficiencies of minus-strand DNA synthesis across different regions of the RNA. Each bar in the graph represents a unique initiation region spanning one to five contiguous nucleotides. The percent conversion of some initiation sites may be underestimated due to polymerization pause sites during plus-strand DNA synthesis (31); incompletely extended plus strands will not be detected by the probe. The numbers on the x axis correspond to positions in HIV-1 NL4-3
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Active and Selective Transcytosis of Cell-Free Human Immunodeficiency Virus through a Tight Polarized Monolayer of Human Endometrial Cells

Active and Selective Transcytosis of Cell-Free Human Immunodeficiency Virus through a Tight Polarized Monolayer of Human Endometrial Cells

Transcytosis of free and cell-associated HIV-1 across a monolayer of epithelial cells. We first investigated whether cell-associated R5- and X4-tropic viruses, as well as the corre- sponding free viral particles, were capable of transcytosis through the HEC-1 monolayer. A significant amount of trans- cytosis was consistently observed in the case of both cell-asso- ciated virus and free virus following contact with the apical membrane of HEC-1 cells at 37°C (Fig. 1A). When performing the experiment at 4°C, we observed that transcytosis of free HIV-1 NDK was inhibited by 90% (Fig. 1B). Virus that was
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Cell-Free Human Immunodeficiency Virus Type 1 Transcytosis through Primary Genital Epithelial Cells

Cell-Free Human Immunodeficiency Virus Type 1 Transcytosis through Primary Genital Epithelial Cells

Cells. TZM-bl cells (generously contributed by John C. Kappes, Xiaoyun Wu, and Tranzyme Inc.) were obtained through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. TZM-bl cells express CD4, CXCR, and CCR5, which render them susceptible to infection, and contain an integrated Escherichia coli lacZ gene driven by the HIV long terminal repeat (59). Upon infection, Tat production from the integrated provirus leads to activation of the lacZ reporter, resulting in synthesis of beta-galactosidase in these cells. Infected cells are identified by enzymatic activity measurement 48 h postinfection, allowing quantitation after a single round of infection as described previously (59). Primary genital epithelial cells (PGECs) were provided by B. Kahn of the Department of Obstetrics and Gynecology at Scripps Clinic. By rotation of cotton swabs against the vaginal walls, several million cells were collected per individual. Cells were immediately placed in sterile phosphate- buffered saline (PBS), held at 4°C, and transported to the laboratory. After centrifugation (300 ⫻ g for 5 min), the cell pellet was digested in 1 mg/ml of collagenase-dispase (Roche Molecular Biochemicals) containing 1 mg/ml of DNase (Sigma) and 0.15 mg/ml of Na-p-tosyl- L -lysine chloromethyl ketone (Sigma) for 1 h at 37°C. The digest was spun down (1,000 ⫻ g for 20 min) and resuspended in 250 mg/ml of PBS-bovine serum albumin (PBS-BSA). After additional centrifugation, the pellet was resuspended in 5 mg/ml of PBS-BSA and loaded onto a 50% Percoll gradient. PGECs were then isolated from con- taminating cells by fluorescence-activated cell sorting (FACS) as previously de- scribed (18) and propagated into collagen type I-coated T-25 flasks in Dulbecco’s modified Eagle medium F12 medium containing 10% fetal calf serum and epi- thelial cell growth supplement (100 ␮ g/ml) (Sigma). As a second source of PGECs, cells were obtained from Whittaker (customized request). PGECs were passaged fewer than three times prior to use in order to maintain their original features.
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